Polypeptide

ABSTRACT

THE INVENTION DESCRIBES A NEW PENTACOSAPEPTIDE OF THE FORMULA. D-SERYL-L-TYROSYL-L-SERYL-L-NORLEUCYL-L-GLUTAMYL-L-HISTIDYL-L-PHENYLALANYL-L-ARGINGYL-L-TRYPTOPHANYLGLYCYL-L-LYSYL-L-PROLYL-L-VALYL-GLYCYL-L-LYSYL-L -LYSYL-LARGINYL-L-ARGINYL-L-PROLYL-L-VALYL-L-HYSYL-L-VALYL-LTYROSYL-L-PROLYL-L-VALINAMIDE, ITS THERAPEUTICALLY ACTIVE ACID ADDITION SALTS AND HEAVY METAL COMPLEXES. THE POLYPEPTIDE OF THE INVENTION, ALSO NAMED D-SER1-NIE4-VALNH2**25A1**25-ACTH, IS PHARMACEUTICALLY USEFUL BECAUSE OF ITS HIGH ADRENOCORITCOTROPIC EFFECT.

United States Patent 3,761,459 POLYPEPTIDE Janos Pless, Basel, Stephan Guttmann, Allschwil, and Roger Boissonnas, Bottmingen, Switzerland, assignors to Sandoz Ltd. (also known as Sandoz A.G.), Basel, Switzerland No Drawing. Filed July 20, 1966, Ser. No. 566,476 Claims priority, application Switzerland, Aug. 24, B65, 11,898/ 65 Int. Cl. C07c 103/52; C07g 7/00 US. Cl. 260-1125 1 Claim ABSTRACT OF THE DISCLOSURE The present invention relates to a new polypeptide and to a process for the production thereof.

The present invention provides the pentacosapeptide of formula D seryl-L-tyrosyl-L-seryl-L-norleucyl-L-glutamyl-L-histidyl-L-phenylalanyl L arginyl-L-tryptophanylglycyl-L-lysyl-L-prolyl-L-valyl-glycyl L lysyl-L-lysyl-L- arginyl L arginyl L prolyl-L-valyl-L-lysyl-L-valyl-L- tyrosyl-L-prolyl-L-valinamide, hereinafter named D-Ser Nle -pentacosapeptide, its therapeutically active acid addition salts and heavy metal complexes. D-Ser -Nle pentacosapeptide, its salts and heavy metal complexes exhibit a high adrenocorticotropic effect.

The synthesis of a pentacosapeptide of formula L-seryl- L-tyrosyl-L-seryl L norleucyl-L-glutamyl-L-histidyl-L- phenylalanyl L arginyl-L-tryptophanyl-glycyl-L-lysyl- L-pr0yl-L-valyl-glycyl-L-lysyl L lysyl L arginyl L- arginyl-L-proyl L valyl L lysyl-L-valyl-L-tyrosyl-L- prolyl-L-valinamide, having a corticotropic effect and hereinafter named Nle -pentacosapeptide, is known.

An advantage of Nle -pentacosapeptide over natural ACTH is that the former has no antigenic effects. A further advantage is that Nle -pentacosapeptide, in contradistinction to natural ACTH, is devoid of a methionine radical in the 4-position, which methionine radical is easily oxidized whereby the hormone becomes inactive; Me -pentacosapeptide contains a norleucine radical in place of the methionine radical present in ACTH, which norleucine radical has the same steric properties as the methionine radical, but is stable to oxidation. Furthermore, Nle -pentacosapeptide has a valinamide radical in the ZS-position, which is not present in this position in natural ACTH. This valinamide radical on the carboxyl terminal protects the peptide chain from enzymatic degradation. However, Nle -pentacosapeptide contains an L-serine radical in the 1-position, as does natural ACTH; this L-serine radical is easily affected by aminopeptidase degradation.

Attempts were therefore made to replace this terminal L-serine radical of Nle -pentacosapeptide by a radical which is stable to aminopeptidase degradation.

ice

Replacement of the terminal L-serine radical of Nle pentacosapeptide by a D-serine radical yielded D-Ser- Nle -pentacosapeptide which is not affected by aminopeptidases. As D-amino acid radicals are not found in the natural, biologically active peptide hormones, it was not to be expected that the replacement of a natural amino acid radical by an antipode not occurring in nature would result in a compound having biological and therapeutical properties not only qualitatively equal but also quantitatively superior to those of natural ACTH, as will be explained in detail hereinafter.

D-Ser -Nle -pentacosapeptide may be produced by methods for the synthesis of compounds of this type in actual use or described in the literature on the subject, it being possible to join together the amino acids in the order indicated in the above formula one at a time or by first forming constituent peptide units and joining these together.

One method of producing D-Ser -Nle -pentacosapeptide consists in that L-valyl-e-N-R-L-lysyl-L-valyl-L-tyrosyl-L- prolyl-L-valinamide, in which R signifies a carbo-tertbutoxy or a carbo-tert-amyloxy, a toluene sulphonyl, a phthalyl, a formyl or a trifiuoroacetyl radical, is condensed with N-carbobenzoxy-L-valyl-glycyl-e-N-R-L-lysyle-N-R-L-lysyl-nitro-L-arginyl-nitro L arginyl-L-proline, in which R has the above significance, the resulting N-carbobenzoxy L valyl-glycyl-e-N-R-L-lysyl-E-N-R-L lysyl-nitro-L-arginyl-nitro L arginyl-L-prolyl-L-valyl-e- N-R-L-lysyl-L-valyl-L-tyrosyl-L-prolyl-L valinamide, in which R has the above significance, is condensed with N-triphenylmethyl-y O tert-butyl L glutamyl-Im-triphenylmethyl L histidyl L phenylalanyl-Larginyl-L- tryptophanyl-glycyl-e-N-R-L-lysyl-L-proline, in which R has the above significance, after removal of the carbobenzoxy radical and the nitro radicals, the resulting N-triphenylmethyl-y O tert-butyl L glutamyl-Im-triphenylrnethyl L histidyl-L-phenylalanyl L arginyl-L- tryptophanyl-glycyl-e-N-R L lysyl L prolyl-L-valylglycyl-e-N-R-L-lysyl-e-N-R-L-lysyl L arginyl-L-arginyl- L-prolyl L valyl-eN-R-L-lysyl-L-valyl L tyrosyl-L- prolyl-L-valinamide, in which R has the above significance, is condensed with N-R'-D-seryl-L-tyrosyl-L-seryl- L-norleucyl azide, in which R signifies 1a triphenylmethyl, a carbo-tert-butoxy or a carbo-tert-amyloxy, a carbobenzoxy, a trifluoroacetyl, an acetyl, a 'chloroacetyl or a formyl radical, after splitting off the N-triphenyhnethyl radical, and all the protective radicals of the resulting new, protected pentacosapeptide N-R-D'-;seryl-Ltyrosyl- L-seryl-L-norleucyl 'y O-tert-butyl-L-glutamyl-Im-triphenylmethyl-L-histidyl-L-phenylalanyl L arginyl L- tryptophanylglycyl-e-N-R-L-lysyl-L-prolyl-L-valyl glycyle N-R-Lelysyl .e N-R-L-lysyl-LarginyLLaarginyl-I: prolyl-L-valyl s-N-R-L-lysyl-L-valyl-L-trosyl-L-prolylvalinamide, in which R and R have the above significance, are removed in one or more stages in an acid medium.

The starting materials for producing D-Ser -Nle pentacosapeptide, insofar as they were hitherto unknown, may be obtained by methods for the synthesis of peptides in actual use or described in the literature, it being possible to join together the amino acids one at a time or by first forming constituents peptide units and joining these together.

It should be noted that D-Ser -Nle -peptacosapeptide may likewise be obtained or used in the form of its salts.

Examples of acids for acid addition salt formation are: acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, salicylic, 2-phenox'yor Z-acetoxy-benzoic, mandelic, methanesulphonic, ethanesulphonic, hydroxyethanesulphonic, benzeneor toluenesulphonic, naphthalenesulphonic and sulphanilic acid, polymeric acids, e.g. tannic, alginic or polygalacturonic acid, polyphloretinic phosphate or carboxymethyl cellulose, and halogen hydracids, e.g. hydrochloric or hydrobromic acid, nitric, thiocyanic, sulphuric and phosphoric acid. Zinc may, for example, be used for the heavy metal complex.

A major advantage of the synthetic pentacosapeptide over the natural hormone extracted from animal material is that the former has no antigenic effects.

According to the usual and accepted standard tests the biological activity of D Ser Nle pentacosapeptide amounts to 625 i130 corticotropin IU for every mg. of free peptide. The pentacosapeptide of the invention was tested in accordance with the third International Standard for corticotropin, which is available in the form of an International Standard for corticotropin and permits the standardization of ACTH preparations in International Units. It has been found that upon intravenous administration the new pentacosaperptide has a longer duration of action than the hitherto known naturally occurring and synthetic ACTH compounds. The dose of the pentacosapeptide of the invention ranges from about 40 to 60 IU daily, in exceptional cases between and 100 IU daily. The unexpectedly high activity of the new pentacosapeptide, which was ascertained upon standardization, has been confirmed upon therapeutic application so that on a weight basis the new pentacosapeptide is more active than all hitherto known naturally occurring and synthetic ACTH compounds.

D-Ser -Nle -pentacosapeptide may be used as medicament, for example in the form of a pharmaceutical preparation. This may contain the said compound in mixture with an organic or inorganic carrier material which is suitable for parenteral administration. Appropriate carrier materials are substances which do not react with the new compound, e.g. gelatin, lactose, starch, magnesium stearate, talcum, vegetable oils, benzyl alcohols, gum arabic, polyalkylene glycols, Valseline, cholesterol and other known pharmaceutical carrier materials. The pharmaceutical preparations may, for example, be used in the liquid form as solutions, suspensions or emulsions. They may be sterilized and/or they may contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents. However, they may contain other therapeutically valuable substances. The new compound may also be administered in the form of a depot preparation as is the case with natural ACTH.

Examples of radicals for blocking the amino radical of the serine radical during the synthetic of the new pentacosapeptide are the triphenylmethyl, the carbo-tertbutoxy and the carbo-tert-amyloxy radical, but other suitable protective radicals, e.g. the carbobenzoxy, the trifluoroacetyl, the acetyl, the chloroacetyl and the formyl radical, may likewise be used. Suitable radicals for blocking the e-amino radical of the lysine radical are the carbotert-butoxy and the carbo-tert-amyloxy radical, but other suitable protective radicals, e.g. the carbobenzoxy, toluenesulphonyl, phthalyl, formyl and trifluoroacetyl radical, may likewise be used. A suitable radical for blocking the 'y-carboxyl radical of the glutaminic acid radical is the tert-butyloxy radical, but other suitable protective radicals, e.g. the methoxy, the ethoxy, the tert-amyloxy, the amide or the benzyloxy radical may likewise be used.

A suitable radical for the blocking of the imidazole radical of the histidine radical is the triphenylmethyl radical, but other suitable protective radicals, e.g. the carbo-tert-butoxy, carbo-tert-amyloxy, carbobenzoxy 0r benzyl radical may likewise be used.

A suitable radical for the blocking of the guanido radical of the arginine radical is the nitro radical, but other suitable protective radicals, e.g. the tosyl, p-nitrocarbobenzoxy or the 2-(isopropyloxycarbonyl)-3,4,5,6-tetrachlorobenzoyl radical may likewise be used. It is also possible to use the protective effect of the protonization of the guanido radical during the synthesis.

The following abbreviations are used in the text and in the drawings:

In the following non-limitative examples all temperatures are indicated in degrees centigrade.

FIG. A.Produetion oi H-Val-Gly-(R)Lys-(R)Lys-Arg-Arg-Pro-Val-(R)Lys-Val-Tyt-Pro-Val-NH;

RRNO:

I I I H al-Gly- Lys-irys-Arg-A rg-P ro-Val-Lys-Val-Tyr-P ro-Val-i-NH;

Fig. B.--Produetion of H-D-Ser-Tyr-Ser-Nle-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Val-NH,

o B O-EOH H-FSer'Nle+0Me G B 0 {Tyr-Ser-NleE-OMe 9 r a Ri-D-Ser-Tyr-Ser-Nlfi-N; H+1u l iis-Phe-Arg-Try-Gly-Iiys-Pro-Val-Gly-llys-i.ys-Arg-Arg-ProNat-hys-Val-Tyr-Pro-Val-i-NH;

H-i-D-Ser-Tyr-Ser-Nle-Glu-His-Phe-Arg-Try-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-.Arg-Pro-Val-Lys-Va1-Tyr-ProVidal-NH:

EXAMPLE 1 peptide is dissolved in hot ethanol and precipitated with l-valyl-glycyl-carbo-tert-butoxy-L- lysyl-carbo-tert-butoxyggg zl g zgfi i fzg gi i g ag l L-lysyl-L-arginyl-L-arginyl-L-prolyl-L-valyl-carbo tert- 2) 'g I y i i a of CTB L LT [a] =36 in dimethyl formamlde, are obtained. ys a y 2 72 g. of CBO-Val-Gly-(CTB)Lyns-(CTB)Lys-(NO 88 g. of CBC-(N0 )Arg-(NO )Arg-Pro-OMe are dis- Arg-(NO )Arg-Pro-Val-(CTB) Lys-Val-Iyr-Pro-Val-IfIH solved in a mixture of 90% dioxane and water and 220 cc. are dissolved in 1.5 litres of 80% acetic acid, a palladium of a 2 N sodium hydroxide solution are added. After catalyst is added, hydrogenation is efiected until hydrogen two hours the solution is diluted with 1 litre of water and is no longer taken up and the catalyst is filtered 01f. After Washed out several times with ethyl acetate. The aqueous r du ing th volume of the solution, the residue is dissolution is subsequently acidified with 4 N hydrochloric solved in 500 ml of methanol, cooled to 5 4.1 g. of acid, the precipitated product is dissolved in a mixture of pl n lph n fi e added a d pr lpltation 1s methanol/acetone (1:1) and precipitation is eifected by subsequently etfeeted wlth ether. 66 g. of H-Val-Glythe addition of ethyl ether. 70 g. of CBO-(NO )Arg- (CTB)Lys (CTB)Lys-Arg-Arg-Pro-Val-(CTB)Lys-Val- (NO )Arg-Pro-0H, having a melting point of 108 (with Tyr-Pro-Yal-NH are obtained as trityl-toluene sulphodecomposition), [oz] '=\-19 in dimethyl formamide, are Hate, haVlIlg a melting point of 185 (WIth deeompos1- obtained. The tripeptide obtained above is dissolved in ]D in dimethyl formemlde- 400 ml. of a 33% solution of hydrogen bromide in glacial acetic acid, the solution is allowed to stand at 20 for EXAMPLE 2 one hour, is concentrated to 200 ml. and precipitated with O tart 1- 1 lq i 1. .h 1- 1. ethyl ether; filtration, washing with ethyl acetate and dryalanyl L p h l. 1 1-N- b ing are effected- 72 of 2) z) butoxy L lysyl-L-prolyl-L-valyl-glycyl-N-carbo-tert- OH'3HBI', having a melting Point Of (with decom' butoxy L-lysyl-N-carbo-tert-butoxy-L-lysyl-L-arginyl- Position), []n in 95% aeetie acid, are L- arginyl-L-prolyl-L-valyl-N-carbo-tert-butoxy-L-lysyltflined- 34 of Y-( Y Y 3 L valyl L-tyrosyl-L-prolyl-L-valinamide. (H-(OTB) (produced from 85 g. of the corresponding hydrazide) are l i i h -A 1 -p 1. added at 0 to a solution of 72 g. of H-(NO )Arg- Gly (CTB)LyS (CTB)Lys-Arg-Arg-PrQ-Val-(CTB) (NO )Arg-Pro-OH hydrobromide in 600 ml. of dimethyl L -v 1. .v 1.

formamide and 56 ml. of triethylamine. The solution is allowed to stand for 16 hours and the solvent is evap- 62 g. of H-Val-Gly-(CTB)Lys-(CTB)Lys-Arg-Argorated. The residue is dissolved in a mixture of n-butanol/ Pro Val-(CTB)Lys-Val-Tyr-Pro-Val-NH 3Tos-OH are ethyl acetate (2:8) and washed several times with dilute 0 dissolved in 300 m1. of pyridine and 300 ml. of acetonisulphuric acid. The volume of the solution is reduced in trile. 57 g. of Trit-(OTB)Glu-(Trit) His-Phe-Arg-Try-Glya vacuum and precipitation is eflected with ether. 90 g. (CTB)Lys-Pro-OH are subsequently added and when of CBO Val Gly-(CTB)Lys-(CTB)Lys-(NO )Argall the material is dissolved, cooling is eifected to 0 and (NO )Arg-Pro-OH, having a melting point of 151 (with 28.6 g. of dicyclohexyl carbodiimide are added. After decomposition), [u] =-38 in methanol, are obtained. shaking at 20 for 24 hours, the urea is filtered off and 56 g. of CBO-Val-Gly-(CTB)Lys-(CTB)Lys-(NO the solution precipitated with ether. The product is dis- Arg-(NO )ArgPro-OH are dissolved in 900 ml. of disolved several times in methanol and precipitated with methyl formamide and 900 ml. of tetrahydrofuran. After ethyl acetate. 90 g. of Trit-(OTB)Glu-(Trit)His-Phe-Argthe addition of 6.2 ml. of triethylamine the solution is Try Gly-(CTB)Lys-Pro-Val-Gly-(C'I'B)Lys-(CTB)Lyscooled to 10 and 4.2 ml. of chloroformic acid ethyl Arg-Arg-Pro-Val-(CTB)Lys-Val-Tyr-Pro-Val-NH tritylester are added at this temperature. After 10 minutes toluene sulphonate, havi g a melting point of 184 (with 36 g. of H-Val-(CTB)Lys-Val-Tyr-Pro-Val-NH in 160 decomposition), [a] =-5 1 in methanol, are obtained. ml. of dimethyl formamide are added and stirring is eifect- 45 g of D 'Y- Y- ed at 20 for a further 16 hours. The solvent is evaporated (CIB)Lys-Pro-Val-Gly-(CTB)Lys-(CTB)Lys-Arg-Arg-Proin a vacuum and the residue washed with water. The Val-(CTB) Lys-Val-Tyr-Pro-Val-NH 3 Tos-OH are dissolved in 50 ml. of 80% acetic acid and the solution is allowed to stand at 30 for 2 hours. 50 ml. of Amberlite IRA-410 in the acetate form are added, filtration and evaporation in a vacuum are effected and the residue is dissolved in methanol. After precipitating with ether, 40 g. of H-(OTB)Glu-(Trit)His-Phe-Arg-Try-Gly-(CTB)Lys-Pro- Val-Gly- (CT B Lys- CTB Lys Arg-Arg-Pro-Val- CTB) Lys-Val-Tyr-Pro-VaI-NH having a decomposition point of 170, [a] =49 in methanol, are obtained.

EXAMPLE 3 L-tyrosyl-L-seryl-L-norleucine methyl ester (H-Tyr-Ser-Nle-OMe) 54 g. of H-Ser-Nle-OMe.HCl and 63 g. of CBO-Tyr- OH are dissolved in 860 ml. of acetonitrile, cooling is effected to 28 ml. of triethylamine are added, cooling is effected to 10 and 41 g. of dicyclohexyl carbodiimide are added. The mixture is stirred at 0 for 16 hours and is then filtered. The precipitate is washed with 1400 ml. of pyridine. The combined filtrates are evaporated and the rsidue crystallized from ethyl acetate. 101 g. of CBO-Tyr-Ser-Nle-OMe, having a melting point of 140- 142, [u] =-15 in dimethyl formamide, are obtained.

51 g. of the resulting product are dissolved in 2 litres of a N solution of HCl in methanol and hydrogznation is effected in the presence of 10 g. of palladium/charcoal. After about 2 hours hydrogen is no longer taken up. Filtration and evaporation are effected and the residue is crystallized from a mixture of mehtanol/ether (3:1). 42 g. of I-ITyr-Ser-Nle-Ome.HCl, having a melting point of 227, [a] =-7 in dimethyl formamide, are obtained.

EXAMPLE 4 Carbo-tert-butoxy-D-seryl-L-tyrosyl-L-seryl-L-norleucine hydrazide. (CTB-D-Ser-Tyr-Ser-Nle-NHNH 60 g. of D-serine methyl ester hydrochloride are dissolved in 200 ml. of dimethyl formamide and 54 ml. of triethylamine, cooling is effected to 0 and the triethylamine hydrochloride is filtered off. Dimethyl formamide is evaporated in a high vacuum and the residue dissolved in 150 ml. of pyridine. 100 g. of tert-butyl-oxy-carbonyl azide are added dropwise and the mixture is allowed to stand at 20 for 2 days. The solvent is evaporated and the product is taken up in ethyl acetate. After washing with water, dilute hydrochloric acid and potasisum bicarbonate solution, drying is effected over sodium sulphate. After evaporating the ethyl acetate, CTB-D-Ser-OMe results as an oil. The ester is dissolved in 500 ml. of methanol and is allowed to stand at 20 with 50 ml. of hydrazine hydrate for 2 days. After evaporating the methanol, the hydrazide crystallizes. After recrystallization from hot ethyl acetate, 53 g. of CTB-D-Ser-NHNH having a melting point of 114, [a] =3 in dimethyl formamide, are obtained.

10 g. of CTB-D-serine hydrazide are dissolved at 10 in 136 ml. of N hydrochloric acid containing 15 g. of sodium chloride. 160 ml. of ethyl acetate and subsequently 3.8 g. of sodium nitrite are added in 3 portions at the same temperature. The mixture is allowed to react for a further 5 minutes at whilst stirring continuously. The ethyl acetate phase is separated, washed with a cold 10% potassium bicarbonate solution and dried with sodium sulphate. A solution of 13 g. of H-Tyr-Ser-Nle-OMe hydrochloride in 60 ml. of dimethyl formamide and 6 ml. of triethylamine is added to the dried solution. The ethyl acetate is subsequently evaporated in a vacuum and the solution allowed to stand at 20 for 16 hours. The remaining solvent is evaporated in a vacuum and the residue dissolved in ethyl acetate. Washing with dilute phosphoric acid and potassium bicarbonate solution and drying over sodium sulphate are effected. After evaporation the solvent and precipitate with ether, g. of CTB-D-Ser-Tyr- Ser-Nle-OMe, having a melting point of 135,

in methanol, are obtained.

11 g. of CTB-D'-Ser-Tyr-Ser-Nle-OMe are dissolved in 100 ml. of methanol and 4.5 ml. of hydrazine hydrate are added. The mixture is allowed to stand over night at 20, whereupon the product crystallizes. Filtration and washing with methanol and petroleum ether are effected. 7.7 g. of CTB-D-Ser-Tyr-Ser-Nle hydrazide, having a melting point of 210, [a] =+6.4 in dimethyl formamide, are obtained.

EXAMPLE 5 D-seryl L tyrosyl-L-seryl-L-norleucyl-L-glutamyl L- histidyl L phenylalanyl-L-arginyl L tryptophanylglycyl L lysyl-L-prolyl-L-valyl-glycyl-L-lysyl-L-lysyl- L-arginyl-L-arginyl L prolyl-L-valyl-L-lysyl-L-valyl- L-tyrosyl-L-prolyl-L-valinamide. (D Ser-Tyr-Ser-Nle- Glu-His-Phe-Arg-Try-Gly Lys-Pro-Val-Glys-Lys-Lys- Arg-Arg-Pro-Val-Lys-Tyr-Pro-Val-NH 2.0 g. of CTB-D-Ser-Tyr-Ser-Nle-NHNH (Example 4) are dissolved in 12 m1. of dimethyl formamide, 4 ml. of water are added, cooling is effected to -10, 2 ml. of 6 N hydrochloric acid and subsequently 280 mg. of sodium nitrite are added, stirring is effected at 5 for 5 minutes, 300 ml. of 0.2 N potassium bicarbonate solution are added and the mixture is centrifuged. The resulting CTB-D-Ser-Tyr-Ser-Nle-N is dissolved in 50 ml. of dimethyl formamide, 10.5 g. of H-Glu(OTB)-(Trit)His- Phe Arg-Try-Gly-(CTB)Lys-Pro-Val-Gly-(CT B) Lys (CTByLys-Arg-Arg-Pro Val (CTB)Lys-Val-Tyr-Pro- Val-NH acetate are added, the mixture is allowed to stand at 0 for 12 hours, a further amount of tetrapeptide azide produced from 2.0 g. CTB D-Ser-Tyr-Ser-Nle- NHNH is added, the mixture is allowed to stand at 0 for 6 hours, is evaporated, treated with ethyl acetate, washed with hot acetone and ethyl acetate and dried in a vacuum. The resulting product is dissolved in 100 ml. of trifiuoroacetic acid, the solution is allowed to stand at 20 in an atmosphere of nitrogen for one hour, is evaporated, treated with ethyl acetate, filtered and dried. The resulting product is dissolved in 500 ml. of 0.2 N acetic acid, the solution is treated with Amberlite IRA- 410 in the acetate form, is filtered and liophilized. After drying over sodium hydroxide, 7.5 g. of H-D-Ser-Tyr-Ser- Nle-Glu-His-Phe-Arg-Try-Gly-Lys-Pro Val-Gly-Lys-Lys- Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Val-NH heptaacetate decahydrate, having a homogeneous behaviour in chromatography and electrophoresis, are obtained. (Total hydrolysis gives the following composition of amino acids:

Microanalysis.-Calculated (percent): C, 51.9; H, 7.5; N, 16.2; 0, 24.4. Found (percent): C, 52.1; H, 7.5; N, 15.6; 0, 24.8. Melting point 209 with decomposition, ]D =80 in N acetic acid.

Galenical Example: Ampoule 25 LU. of peptide (mg).

G. Concentrated acetic acid about 0.0011 Sodium acetate.3H O 0.002 Sodium chloride 0.007 Benzyl alcohol 0.009

Water ad 1.005 g.

What is claimed is:

1. D-seryl-L-trosyl-L-seryl-L-norleucyl L glutamyl- L histidyl L phenylalanyl-L-arginyl-L-tryphophanylglycyl L lysyl-L-prolyl-L-valyl-glycyl-L-lysyl-L-lysyl- L-arginyl L arginyl-L-prolyl-L-valyl-L-lysyl-L-valyl-L- tyrosyl L prolyl-L-valinamide and the pharma'ceutically 10 acceptable acid addition salts and heavy metal complexes Guttmann et al.: Acta Acad. Sci. Hung 44, 141-142 thereof. (1965 References Cited Jenny 8t 31.: Experientia 22, 528530 Netherlands Application, 6,510,560, February 1966, UNITED STATES PATENTS I 5 Ciba (assignee). 3,228,926 1/1966 Kappeler et al. 260112.5

OTHER REFERENCES ELBERT L. ROBERTS, Primary Examiner Bolssonnas et al.: Experientia 22, 526 (1966). US. Cl. X.R.

Doepfner: Experientia 22, 527528 (1966). 10 424179 

